Mouse mammary tumour virus (MMTV) is a retrovirus that is associated with mammary tumorigenesis in susceptible mice (Salmons, B. and Gxc3xczburg, W. H., Virus Res., 8:81-102, 1987). The virus is transmitted from the mother mouse to the suckling offspring via the milk. In addition to the usual retroviral genes gag, pol and env, the Long Terminal Repeat (LTR) of Mouse Mammary Tumour Virus (MMTV) contains an open reading frame (ORF) (Donehower, L. A. et al., J. Virol., 37:226-238, (1981); Kennedy, N. et al., Nature, 295:622-624 (1982)) which is highly conserved between different MMTV isolates (Brandt-Carlson, C. et al., Virology, 193:171-185 (1993)). Although ORF specific transcripts have yet to be cloned, in part due to their low abundance, a splice acceptor site has been mapped immediately upstream of the 3xe2x80x2 LTR which is presumed to generate putative 1.7 kb ORF transcripts (Wheeler, D. A., et al., J. Virol., 46:42-49 (1983); van Ooyen, A. J. et al., J. Virol., 46:362-370 (1983)). Recently, a novel promoter has been identified in the MMTV 5xe2x80x2LTR and transcripts initiating from this promoter also splice to the ORF acceptor site (Gxc3xczburg, W. H. et al., Nature, 364:154-158 (1993)), increasing the potential for diversity of ORF related products.
Two biological activities, defined by functional assays, have been ascribed to products of the ORF. One of these activities is a transcriptional repressor, Naf, which downregulates in trans expression from MMTV based constructs (Salmons, B., et al., J. Virol., 64:6355-6359, (1990); Gxc3xczburg, W. H. and Salmons, B., Biochem. J., 283:625-632 (1992)). The second activity displayed by the MMTV ORF is a superantigen. (Sag) activity (Choi, Y., et al., Nature, 350:203-207 (1991); Acha-Orbea, H., et al., Nature, 350:207-211 (1991)). Expression of Sag in vivo results in the stimulation and growth, followed by deletion, of reactive T cells (reviewed in Acha-Orbea, H. and MacDonald, H. R., Trends in Microbiology, 1:32-34 (1993)). This effect is specific in that the Sag of a given MMTV variant interacts with specific classes of the twenty described V13 chains of the T cell receptor (Pullen, A. M., et al., J. Exp. Med., 175:41-47 (1992), Huber, B. T., Trends in Genetics, 8:399-402 (1992)).
The viral Sag has been shown to be a type II membrane anchored glycoprotein of 45 KDa by in vitro translation studies (Korman, A. J., et al., The EMBO J., 11:1901-1905 (1992), Knight, A. M., et al., Eur. J. Immunol., 175:879-882 (1992)). Further, Sag proteins of 45/47 kDa have also been synthesized in baculovirus (Brandt-Carlson, C. and Butel, J. S., J. Virol., 65:6051-6060 (1991); Mohan, N. et al., J. Exp. Med., 177:351-358 (1993)) and vaccinia virus (Krummenacher, C. and Diggelmann, H., Mol. Immunol., 30:1151-1157 (1993)) expression systems. This 45/47 kDa glycoprotein may require processing to a 18 kDa cleavage product (Winslow, G. M. et al., Cell, 71:719-730 (1992)). A Sag specific monoclonal antibody detects Sag expression on LPS-activated, but not nonstimulated, B cells even though the latter cells express a functional Sag. Thus undetectable levels of Sag are sufficient for superantigen activity ((Winslow, G. M. et al., Cell, 71:719-730 (1992); Winslow, G. M. et al., Immunity, 1:23-33 (1994)).
The use of retroviral vectors (RV) for gene therapy has received much attention and currently is the method of choice for the transferral of therapeutic genes in a variety of approved protocols both in the USA and in Europe (Kotani, H., et al., Human Gene Therapy, 5:19-28 (1994)). However, most of these protocols require that the infection of target cells with the RV carrying the therapeutic gene occurs in vitro, and successfully infected cells are then returned to the affected individual (Rosenberg, S. A., et. al., Human Gene Therapy, 3:75-90 (1992), Anderson, W. F., Science, 256:808-813 (1992)). Such ex vivo gene therapy protocols are ideal for correction of medical conditions in which the target cell population can be easily isolated (e.g., lymphocytes). Additionally the ex vivo infection of target cells allows the administration of large quantities of concentrated virus which can be rigorously safety tested before use.
Unfortunately, only a fraction of the possible applications for gene therapy involve target cells that can be easily isolated, cultured and then reintroduced. Additionally, the complex technology and associated high costs of ex vivo gene therapy effectively preclude its disseminated use world-wide. Future facile and cost-effective gene therapy will require an in vivo approach in which the viral vector, or cells producing the viral vector, are directly administered to the patient in the form of an injection or simple implantation of RV producing cells.
This kind of-in vivo approach-, of course, introduces a variety of new problems. First of-all, and above all, safety consideration have to be addressed. Virus will be produced, possibly from an-implantation of virus producing cells, and there will be-no opportunity to precheck the produced virus. It is important to be aware of the finite risk involved in the use of such systems, as well as trying to produce new systems that minimize this risk. The essentially random integration of the proviral form of the retroviral genome into the genome of the infected cell led to the identification of a number of cellular proto-oncogenes by virtue of their insertional activation (Varmus, H. xe2x80x9cRetrovirusesxe2x80x9d, Science, 240:1427-1435 (1988)). The possibility that a similar mechanism may cause cancers in patients treated with RVs carrying therapeutic genes intended to treat other preexistent medical conditions, has posed a recurring ethical problem. Most researchers would agree that the probability of the replication defective RV, such as all those currently used, integrating. into or near a cellular gene involved in controlling cell proliferation is vanishingly small. However, it is generally also assumed that the explosive expansion of a population of replication competent retrovirus from a single infection event, will eventually provide enough integration events to make such a phenotypic integration a very real possibility.
Retroviral vector systems are optimized to minimize the chance of replication competent virus being present. However, it has been well documented that recombination events between components of the RV system can lead to the generation of potentially pathogenic replication competent virus and a number of generations of vector systems have been constructed to minimize the risk of recombination (Salmons, B. and Gxc3xcnzburg, W. H., Human Gene Therapy, 4:129-141 (1993)). However, little is known about the finite probability of these events. Since it will never be possible to reduce the risk associated with this or other viral vector systems to zero, an informed risk-benefit decision will always have to be taken. Thus it becomes very important to empirically: determine the chance of (Donehower, L. A. et al., J. Virol., 37:226-238, (1981)) insertional disruption or activation of single genes by retrovirus integration and (Kennedy, N. et al., Nature, 295:622-624 (1982)) the risk of generation of replication competent virus by recombination in current generations of packaging cell lines. A detailed examination of the mechanism by which these events occur will also allow the construction of new types of systems designed to limit these events.
A further consideration for practical in vivo gene therapy, both from safety considerations as well as from an efficiency and from a purely practical point of view, is the targeting of RVs. It is clear that therapeutic genes carried by vectors should not be indiscriminately expressed in all tissues and cells, but rather only in the requisite target cell. This is especially important if the genes to be transferred are toxin genes aimed at ablating specific tumour cells. Ablation of other, nontarget cells would obviously be very undesirable. Targeting of the expression of carried therapeutic genes can be achieved by a variety of means.
Retroviral vector systems consist of two components:
1. the retroviral vector itself is a modified retrovirus (vector plasmid) in which the genes encoding for the viral proteins have been replaced by therapeutic genes optionally including marker genes to be transferred to the target cell. Since the replacement of the genes encoding for the viral proteins effectively cripples the virus it must be rescued by the second component in the system which provides the missing viral proteins to the modified retrovirus.
The second component is:
2. a cell line that produces large quantities of the viral proteins, however lacks the ability to produce replication competent virus. This cell line is known as the packaging cell line and consists of a cell line transfected with a second,plasmid carrying the genes enabling the modified retroviral vector to be packaged. This plasmid directs the synthesis of the necessary viral proteins required for virion production.
To generate the packaged vector, the vector plasmid is transfected into the packaging cell line. Under these conditions the modified retroviral genome including the inserted therapeutic and optional marker genes is transcribed from the vector plasmid and packaged into the modified retroviral particles (recombinant viral particles). A cell infected with such a recombinant viral particle cannot produce new vector virus since no viral proteins are present in these cells. However, the vector carrying the therapeutic and marker genes is present and these can now be expressed in the infected cell.
The retroviral genome consists of an RNA molecule with the structure R-U5-gag-pol-env-U3-R (FIG. 6). During the process of reverse transcription, the U5 region is duplicated and placed at the right hand end of the generated. DNA molecule, whilst the U3 region is duplicated and placed at the left hand end of the generated DNA molecule (FIG. 6). The resulting structure U3-R-U5 is called LTR (Long Terminal Repeat) and is thus identical and repeated at both ends of the DNA structure or provirus. The U3 region at the left hand end of the provirus harbours the promoter (see below). This promoter drives the synthesis of an RNA transcript initiating at the boundary between the left hand U3 and R regions and terminating at the boundary between the right hand R and U5 region (FIG. 6). This RNA is packaged into retroviral particles and transported into the target cell to be infected. In the target cell the RNA genome is again reverse transcribed as described above.
According to the procon principle a retroviral vector is constructed in which the right hand U3 region is altered (FIG. 7), but the normal left hand U3 structure is maintained (FIG. 7); the vector can be normally transcribed into RNA utilizing the normal retroviral promoter located within the left hand U3 region (FIG. 7). However, the generated RNA will only contain the altered right hand U3 structure. In the infected target cell, after reverse transcription, this altered U3 structure will be placed at both ends of the retroviral structure (FIG. 7).
If the altered region carries a polylinker (see below) instead of the U3 region then any promoter, including those directing tissue specific expression (see below) can be easily inserted. This promoter will then be utilized exclusively in the target cell for expression of linked genes carried by the retroviral vector. Alternatively or additionally DNA segments homologous to one or more cellular sequences can be inserted into the polylinker for the purposes of gene targeting.
In the packaging cell line the expression of the retroviral vector is regulated by the normal unselective retroviral promoter (FIG. 7). However, as soon as the vector enters the target cell promoter conversion occurs, and the therapeutic genes are expressed from a tissue specific promoter of choice introduced into the polylinker (FIG. 7). Not only can virtually any tissue specific promoter be included in the system, providing for the. Selective targeting of a wide variety of different cell types, but additionally, following the conversion event, the structure and properties of the retroviral vector no longer resembles that of a virus. This, of course, has extremely important consequences from a safety point of view, since ordinary or state of the art retroviral vectors readily undergo genetic recombination with the packaging vector to produce, potentially pathogenic viruses. Promoter conversion (Procon) vectors do not resemble retroviruses because they no longer carry U3 retroviral promoters after conversion thus reducing-the possibility of genetic recombination.
It is an object of the present invention to provide novel usages for the nucleotide and amino acid sequences comprising Naf activity.
It is a further object of the present invention to provide novel usages for the nucleotide and amino acid sequences comprising Sag activity.
It is also a further object of the present invention to provide novel vectors useful for gene therapy of viral infections.
It is still a further object of the present invention to provide novel vectors useful for gene therapy of diseases associated with B cells.
According to one aspect of the present invention there is provided a novel usage of a nucleotide sequence or amino acid sequence of a derivative thereof comprising Naf activity for repressing the expression of viral promoters, e.g., for the treatment of viral infections.
In another aspect the invention provides a novel recombinant DNA vector for introducing into an eucaryotic cell DNA for repressing the expression of heterologous viral promoters, the vector comprising, in operable linkage, a) the DNA of or corresponding to at least a portion of a vector, which portion is capable of infecting and directing the expression in the target cells; and b) one or more coding sequences wherein at least one sequence encodes for a peptide (protein) with Naf activity or a derivative thereof. Optionally, the recombinant vector of the present invention can include at least one sequence encoding a therapeutic and/or non-therapeutic peptide (protein). For example, the peptide (protein) can be (xcex2-galactosidase, neomycin, alcohol dehydrogenase, puromycin, hypoxanthine phosphoribosyl transferase (HPRT), hygromycin, secreted alkaline phosphatase, Herpes Simplex Virus thymidine kinase, cytosine deaminase, guanine phosphoribosyl transferase (gpt), cytochrome P 450, cell cycle regulatory genes which codes for proteins including P.T.O. or SDI, tumor supressor gene which codes for proteins including p53, antiproliferation genes which codes for proteins including melittin and cecropin, or genes which codes for cytokines) such as IL-2.
Said vector is selected from the group of viral and plasmid vectors. In particular said viral vector is selected from the group of RNA and DNA viruses. Said plasmid vector is preferably selected from the group of eucaryotic expression vectors and wherein said RNA virus vector is selected from retrovirus vectors. Said DNA virus is preferably selected from the group of adenoviruses, adenovirus associated viruses and herpes viruses; and wherein said retroviral vector is preferably selected from the group of procon vectors. In a preferred embodiment the retroviral genome is replication-defective.
In one embodiment the present invention uses the principle of promoter conversion typical for retroviruses.
The procon vector includes preferably, in operable linkage, a 5xe2x80x2LTR region; one or more of said coding sequences wherein at least one sequence encodes for a peptide with Naf activity or a derivative thereof for repressing the expression of heterologous viral promoters; and a 3xe2x80x2LTR region; said 5xe2x80x2LTR region comprising the structure U3-R-U5 and said 3xe2x80x2LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region to undergo promoter conversion.
In a further preferred embodiment, the retrovirus vector includes, in operable linkage, a 5xe2x80x2LTR region and a 3xe2x80x2LTR region, said 5xe2x80x2LTR region comprising the structure U3-R-U5 and said 3xe2x80x2LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by one or more of said coding sequences wherein at least one sequence encodes for a peptide with Naf activity expressed from either the viral or a heterologous promoter for repressing the expression of heterologous viral promoters followed by the R and U5 region.
With reference to the procon vectors,.said polylinker sequence carries at least one unique restriction site and contains preferably at least one insertion of a heterologous DNA fragment. Said heterologous DNA fragment is preferably selected from regulatory elements and promoters, preferably being target cell specific in their expression.
For a complete disclosure of the procon vectors, the content of the Danish application DK1017/94, filed on Sep. 2, 1994 is completely included within the present application or incorporated herein by reference.
The recombinant DNA vectors provided by the present invention may preferably be used to treat viral infections by repressing viral promoters.
The recombinant DNA vectors provided in the present invention may be preferably used to repress heterologous viral promoters selected from HIV or MLV promoters.
In a further aspect the invention provides a novel usage of a nucleotide sequence or amino acid sequence or a derivative thereof comprising Sag activity in the gene therapy of disorders associated with B or T cells.
In a preferred embodiment, a recombinant DNA vector for introducing into a B or T cell DNA for gene therapy of disorders associated with B or T cells is provided, comprising, in operable linkage,
a) the DNA of or corresponding to at least a portion of a vector, which portion is capable of infecting and directing the expression in the B or T cells; and
b) one or more coding sequences wherein at least one sequence encodes for a peptide with Sag activity or a derivative thereof and at least one sequence encodes for a therapeutic peptide or protein.
Said vector is selected from the group of viral and plasmid vectors. In particular said viral vector is selected from the group of RNA and DNA viruses. Said plasmid vector is preferably selected from the group of eucaryotic expression vectors and wherein said RNA virus vector is selected from retrovirus vectors. Said DNA virus is preferably selected from the group of adenoviruses, adenovirus associated viruses and herpes viruses; and wherein said retroviral vector is preferably selected from the group of procon vectors. In a preferred embodiment the retroviral genome is replication-defective.
In a preferred embodiment said procon vector includes, in operable linkage, a 51xe2x80x2LTR region; one or more of said coding sequences wherein at least one sequence encodes for a peptide with Sag activity or a derivative thereof and at least one sequence encodes for a therapeutic peptide; and a 3xe2x80x2LTR region; said 5xe2x80x2LTR region comprising the structure U3-R-U5 and said 3xe2x80x2LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by a polylinker sequence, followed by the R and U5 region to undergo promoter conversion.
According to a further preferred embodiment a retrovirus vector is used which includes, in operable linkage, a 5xe2x80x2LTR region and a 3xe2x80x2LTR region, said 5xe2x80x2LTR region comprising the structure U3-R-U5 and said 3xe2x80x2LTR region comprising a completely or partially deleted U3 region wherein said deleted U3 region is replaced by one or more of said coding sequences wherein at least one sequence encodes for a peptide with Sag activity or a derivative thereof and at least one sequence encodes for a therapeutic peptide (protein) expressed from either the viral or a heterologous promoter, followed by the R and U5 region.
Gene expression is regulated by promoters. In the absence of promoter function a gene will not be expressed. The normal MLV retroviral promoter is fairly unselective in that it is active in most cell types. However, a number of promoters exist that show activity only in very specific cell types. Such tissue-specific promoters will be the ideal candidates for the regulation of gene expression in retroviral vectors,.limiting expression of the therapeutic genes to specific target cells.
The target cell specific regulatory elements and promoters are preferably, but not limited, selected from one or more elements of the group consisting of HIV, Whey Acidic Protein (WAP), Mouse Mammary Tumour Virus (MMTV), xcex2-lactoglobulin and casein specific regulatory elements and promoters, which may be used to target human mammary tumours, pancreas specific regulatory elements and promoters including carbonic anhydrase II and xcex2-glucokinase regulatory elements and promoters, lymphocyte specific regulatory elements and promoters including immunoglobulin and MMTV lymphocytic specific regulatory elements and promoters and MMTV specific regulatory elements and promoters conferring responsiveness to glucocorticoid hormones or directing expression to the mammary gland, T-cell specific regulatory elements and promoters such as T-cell receptor gene and CD4 receptor promoter and B-cell specific regulatory elements and promoters such as immunoglobulin promoter or mb1. Said regulatory elements and promoters regulate preferably the expression of at least one of the coding sequences of said retroviral vector.
The LTR regions are preferably, but not limited, selected from at least one element of the group consisting of LTRs of Murine Leukaemia Virus (MLV), Mouse Mammary Tumour Virus (MMTV), Murine Sarcoma Virus (MSV), Simian Immunodeficiency Virus (SIV), Human Immunodeficiency Virus (HIV), Human T-cell Leukaemia Virus (HTLV), Feline Immunodeficiency Virus (FIV), Feline Leukaemia Virus (FELV) Bovine Leukaemia Virus (BLV) and Mason-Pfizer-Monkey virus (MPMV).
The Naf or Sag encoding sequences of the present invention will be placed under the transcriptional control of, for instance, the HIV promoter or a minimal promoter placed under the regulation of the HIV tat responsible element (TAR) to target HIV infected cells. Targeting will be achieved because the HIV promoter is dependent upon the presence of Tat, an HIV encoded autoregulatory protein (Haseltine, W. A., FASEB J., 5:2349-2360 (1991)).
Thus only cells infected with HIV and therefore expressing Tat will be able to produce the Naf or Sag peptide encoded by the vector. Alternatively, the Naf or Sag peptide could be expressed from T cell specific promoters such as that from the CD4 or T cell receptor gene. In order to target tumour cells, promoters from genes known to be overexpressed in these cells (for example c-myc, c-fos) may be used.
The Naf or Sag encoding sequences of the present invention may be placed also under the transcriptional control of other promoters known in the art. Examples for such promoters are of the group of SV40, cytomegalovirus, Rous sarcoma virus, xcex2-actin, HIV-LTR, MMTV-LTR, target cell specific promoters, B or T cell specific promoters and tumour specific promoters.
In one embodiment of the invention the Naf or Sag peptide is expressed from MMTV promoters such as the MMTVP2 promoter (Gxc3xczburg, W. H., et. al., Nature, 364:154-158 (1993)).
The retroviral vector is in one embodiment of the invention a BAG vector (Price, J. D., et. al., Proc. Natl. Acad. Sci. USA, 84:156-160 (1987)), but includes also other retroviral vectors.
According to a preferred embodiment of the invention at least one retroviral sequence encoding for a retroviral protein involved in integration of retroviruses is altered or at least partially deleted.
The vector preferably contains DNA fragments homologous to one or more cellular sequences. The regulatory-elements and promoters are preferably regulatable by transacting molecules.
In a further embodiment of the invention a retroviral vector system is provided comprising a retroviral vector as described above as a first component and a packaging cell line harbouring at least one retroviral or recombinant retroviral construct coding for proteins required for said retroviral vector to be packaged.
The packaging cell line harbours retroviral or recombinant retroviral constructs coding for those retroviral vector. The packaging cell line is preferably selected from an element of the group consisting of "psgr"2, "psgr"-Crip, "psgr"-AM, GP+E-86, PA317 and GP+envAM-12.
After replicating the retroviral vector of the invention as described above in a retroviral vector system as described above, a retroviral provirus is provided wherein U3 or said polylinker and any sequences inserted in said polylinker in the 3xe2x80x2LTR become duplicated during the process of reverse transcription in the infected target cell and appear in the 5xe2x80x2LTR as well as in the 3xe2x80x2LTR of the resulting provirus, and the U5 of 5xe2x80x2LTR become duplicated during reverse transcription and appear at the 3xe2x80x2LTR as well as in the 3xe2x80x2LTR of the resulting provirus.
According to the invention the term xe2x80x9cpolylinkerxe2x80x9d is used for a short stretch of artificially synthesized DNA which carries a number of unique restriction sites allowing the easy insertion of any promoter or DNA segment. The term xe2x80x9cheterologousxe2x80x9d is used for any combination of DNA sequences that is not normally found intimately associated in nature. The retroviral vector of the invention refers to a DNA sequence retroviral vector on the DNA sequence level.
The invention includes, however, also mRNA of a retroviral provirus according to the invention and any RNA resulting from a retroviral vector according to the invention and cDNAs thereof.
A further embodiment of the invention provides non-therapeutical or therapeutical method for introducing Naf or Sag sequences into human or animal cells in vitro and in vivo comprising transfecting a packaging cell line of a retroviral vector system according to the invention with a retroviral vector according to the invention and infecting a target cell population with recombinant retroviruses produced by the packaging cell line.
The retroviral vector, the retroviral vector system and the retroviral provirus as well as RNA thereof may be used for producing a pharmaceutical composition for somatic gene therapy in mammals including humans. Furthermore, they are used for targeted integration in homologous cellular sequences.
The retroviral promoter structure is termed LTR. LTR""s carry signals that allow them to jump in and out of the genome of the target cell. Such jumping transposable elements can also contribute to pathogenic changes. Retroviral vectors vectors can carry modified LTRs that no longer carry the signals required for jumping. Again this increases the potential safety of these vector systems.
Further objects, features and advantages will be apparent from the following description of preferred embodiments of the invention.